The present invention relates to pathway engineering and in particular to biocatalytic methods for the production of ascorbic acid intermediates. In particular, the invention provides methods for the production of ascorbic acid intermediates in non-fermentative systems.
L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical and food industry as a vitamin and antioxidant. The synthesis of ASA has received considerable attention over many years due to its relatively large market volume and high value as a specialty chemical. The Reichstein-Grussner method, a chemical route from glucose to ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328). Lazarus et al. (1989, xe2x80x9cVitamin C: Bioconversion via a Recombinant DNA Approachxe2x80x9d, Genetics and Molecular Biology of Industrial Microorganisms, American Society for Microbiology, Washington D.C. Edited by C. L. Hershberger) disclosed a bioconversion method for production of an intermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically converted to ASA. This bioconversion of carbon source to KLG involves a variety of intermediates, the enzymatic process being associated with co-factor dependent reductase activity. Enzymatic co-factor regeneration involves the use of enzymes to regenerate co-factors such as NAD+ to NADH or NADP+ to NADPH at the expense of another substrate that is then oxidized.
There remains a need for economically feasible methods for the production of ASA intermediates. In particular, when such methods involve the use of enzymatic activities which require co-factor, it would be particularly desirable to have methods which provide for co-factor regeneration. The present invention addresses that need.
The present invention relates to the non-fermentative production of ASA intermediates, e.g., KDG, DKG and KLG, and ultimately their conversion to end products, e.g., erythorbate and ascorbic acid, from a carbon source in a biocatalytic environment. See FIG. 2 for a schematic representation of the production of these intermediates and products.
The present invention also relates to a non-fermentative process for the production of ASA intermediates wherein required co-factor is regenerated. The biocatalytic environment may comprise viable or non-viable host cells which contain at least one enzymatic activity capable of processing the carbon source to the desired intermediate.
When KDG is the desired ASA intermediate, the bioreactor is provided with a carbon source which is biocatalytically converted through at least one oxidative step to KDG. In this embodiment, the host cell may comprise a mutation(s) in a gene encoding an oxidative enzymatic activity specific to oxidizing the KDG. When DKG is the desired ASA intermediate, the bioreactor is provided with a carbon source which is biocatalytically converted through at least one oxidative step to DKG. Depending upon the host cell used, the host cell may comprise a mutation(s) in a gene encoding an oxidizing or reducing enzymatic activity such that DKG is not further converted to other intermediates. When KLG is the desired ASA intermediate, the bioreactor is provided with a carbon source which is biocatalytically converted through at least one oxidative step and at least one reducing step to KLG. Depending upon the host cell used, the host cell may comprise a mutation(s) in a gene encoding an oxidizing or reducing enzymatic activity such that KLG is not further converted to other intermediates. When the oxidative step and reducing step require co-factor, the method provides a means for co-factor regeneration. Therefore, the present invention is based, in part, upon the discovery that catalytic amounts of co-factor can be regenerated in a non-fermentative, or in vitro, method for the production of KLG from a carbon source.
The host cells may be recombinant comprising at least one heterologous enzymatic activity. The process may be performed as a batch process or a continuous process. The host cells are preferably members of the family Enterobacteriacea and in one embodiment, the member is a Pantoea species and in particular, Pantoea citrea. Pantoea citrea can be obtained from ATCC having ATCC accession number 39140, for example.
The host cells may be lyophilized, permeabilized, or otherwise treated to reduce viability or mutated to eliminate glucose utilization for cell growth or metabolism as long as the enzymatic activity is available to convert the carbon source to the desired intermediate. The intermediates may be further processed to the end products of erythorbate or ASA.
Accordingly, in one aspect, the present invention provides a method for the production of the intermediate DKG or KDG from a carbon source comprising enzymatically oxidizing the carbon source by at least one oxidative enzymatic activity to yield DKG or KDG. In another embodiment, the process comprises oxidizing the carbon source by a first oxidative enzymatic activity to yield a first oxidative product and oxidizing said first oxidative product by a second oxidative enzymatic activity to yield KDG. In one embodiment, the first oxidative enzymatic activity is a GDH activity and the second oxidative enzymatic activity is a GADH activity. KDG may be further converted to erythorbate. The process may further comprise oxidizing KDG by a third oxidative enzymatic activity to yield DKG.
For production of KLG, if the carbon source is KDG, the method comprises the steps of enzymatically oxidizing the KDG by at least one oxidative enzymatic activity to an oxidation product; and enzymatically reducing said oxidation product by at least one reducing enzymatic activity to 2-KLG. Alternatively, if DKG is the carbon source, DKG is converted to KLG by a reducing enzymatic activity.
In one embodiment, at least one oxidative enzymatic activity is bound to host cell membranes and in another embodiment, at least one oxidative enzymatic activity is in solution and in another embodiment, at least one enzymatic activity is immobilized. In the process for producing KDG, it is preferred that the host cell comprises a mutation in the nucleic acid encoding a KDGDH activity, such that the KDG is not further oxidized.
The present invention also provides a process for the non-fermentative production of 2-KLG from a carbon source, wherein said process comprises the following steps in any order, enzymatically oxidizing the carbon source by at least one oxidative enzymatic activity to an oxidation product; and enzymatically reducing said oxidation product by at least one reducing enzymatic activity to 2-KLG. In one embodiment, the carbon source is KDG. In another embodiment, said oxidative enzymatic activity requires an oxidized form of an enzymatic co-factor and said reducing enzymatic activity requires a reduced form of said enzymatic co-factor and said oxidized form of said co-factor and said reduced form of said co-factor are recycled between at least one oxidizing step and at least one reducing step.
In another embodiment, the process comprises the following steps in any order: enzymatically oxidizing the carbon source by a first oxidative enzymatic activity to a first oxidation product; enzymatically oxidizing the first oxidation product by a second oxidative enzymatic activity to a second oxidation product; enzymatically oxidizing the second oxidation product by a third oxidative enzymatic activity to a third oxidation product; and enzymatically reducing the third oxidation product by a reducing enzymatic activity to 2-KLG. In one embodiment, at least one of said first, second and third oxidative enzymatic activities requires an oxidized form of an enzymatic co-factor and said reducing enzymatic activity requires a reduced form of said enzymatic co-factor and wherein said oxidized form of said co-factor and said reduced form of said co-factor are recycled between at least one oxidizing step and the reducing step. In one embodiment of the process, the first oxidative enzymatic activity requires an oxidized form of said enzymatic co-factor.
In one embodiment of the process, the carbon source is glucose and said first oxidative enzymatic activity is a glucose dehydrogenase activity. The glucose dehydrogenase activity may be obtained from a bacterial, yeast or fungal source, including T. acidophilum, Cryptococcus uniguttalatus and Bacillus species. In another embodiment, each of said first, said second enzyme and said third enzymatic activities is a dehydrogenase activity. In one embodiment, at least one of said first, said second, said third and said fourth enzyme activities are immobilized, in another at least one is in solution and in another at least one is bound to the membrane of a viable or non-viable host cell.
In a further embodiment, the second oxidative enzymatic activity is a GADH and the third oxidative enzymatic activity is KDGDH. In another embodiment, the reductase activity is obtainable from a bacterial, yeast or fungal source and in a preferred embodiment is 2,5 DKG reductase.
In yet another embodiment of the method, the first oxidation product is gluconate, the second oxidation product is 2-KDG, and the third oxidation product is 2,5-DKG.
In a further embodiment of the process, the recombinant host cell has a mutation of at least one naturally occurring dehydrogenase activity and is preferably a deletion in the naturally occurring membrane bound GDH activity. The host cell may further comprise nucleic acid encoding a heterologous GDH activity.
Some embodiments of the process will proceed in a manner allowing for enzymatic co-factor recycling. In one aspect, the oxidized form of the enzymatic cofactor is NADP+ and the reduced form of said enzymatic cofactor is NADPH. In another aspect, the oxidized form of said enzymatic cofactor is NAD+ and the reduced form is NADH. Other co-factors useful in the process of the present invention include ATP, ADP, FAD and FMN.
In one embodiment, the process proceeds in an environment comprising organic solvents and in another, the process proceeds in an environment comprising long polymers.
The present invention also provides vectors and recombinant host cells comprising enzymatic activities which are used in the methods for producing the ASA intermediates. In one embodiment, the host cell comprises heterologous nucleic acid encoding GDH obtainable from species including including T. acidophilum, Cryptococcus uniguttalatus and Bacillus species and/or DKG reductase obtainable from Corynebacterium or Erwinia.